Tamar Kapanadze

Regulation of accumulation and function of myeloid derived suppressor cells in different murine models of hepatocellular carcinoma

BACKGROUND & AIMS Myeloid derived suppressor cells (MDSC) are immature myeloid cells with immunosuppressive activity. They accumulate in tumor-bearing mice and humans with different types of cancer, including hepatocellular carcinoma (HCC). The aim of this study was to examine the biology of MDSC in murine HCC models and to identify a model, which mimics the human disease. METHODS The comparative analysis of MDSC was performed in mice, bearing transplantable, diethylnitrosoamine (DEN)-induced and MYC-expressing HCC at different ages. RESULTS An accumulation of MDSC was found in mice with HCC irrespective of the model tested. Transplantable tumors rapidly induced systemic recruitment of MDSC, in contrast to slow-growing DEN-induced or MYC-expressing HCC, where MDSC numbers only increased intra-hepatically in mice with advanced tumors. MDSC derived from mice with subcutaneous tumors were more suppressive than those from mice with DEN-induced HCC. Enhanced expression of genes associated with MDSC generation (GM-CSF, VEGF, IL6, IL1β) and migration (MCP-1, KC, S100A8, S100A9) was observed in mice with subcutaneous tumors. In contrast, only KC levels increased in mice with DEN-induced HCC. Both KC and GM-CSF overexpression or anti-KC and anti-GM-CSF treatment controlled MDSC frequency in mice with HCC. Finally, the frequency of MDSC decreased upon successful anti-tumor treatment with sorafenib. CONCLUSIONS Our data indicate that MDSC accumulation is a late event during hepatocarcinogenesis and differs significantly depending on the tumor model studied.

Comparative analysis of monocytic and granulocytic myeloid-derived suppressor cell subsets in patients with gastrointestinal malignancies

Myeloid-derived suppressor cells (MDSC) are a heterogenous population of cells comprising myeloid progenitor cells and immature myeloid cells, which have the ability to suppress the effector immune response. In humans, MDSC have not been well characterized owing to the lack of specific markers, although it is possible to broadly classify the MDSC phenotypes described in the literature as being predominantly granulocytic (expressing markers such as CD15, CD66, CD33) or monocytic (expressing CD14). In this study, we set out to perform a direct comparative analysis across both granulocytic and monocytic MDSC subsets in terms of their frequency, absolute number, and function in the peripheral blood of patients with advanced GI cancer. We also set out to determine the optimal method of sample processing given that this is an additional source of heterogeneity. Our findings demonstrate consistent changes across sample processing methods for monocytic MDSC, suggesting that reliance upon cryopreserved PBMC is acceptable. Although we did not see an increase in the population of granulocytic MDSC, these cells were found to be more suppressive than their monocytic counterparts.

Anti‐Gr‐1 antibody depletion fails to eliminate hepatic myeloid‐derived suppressor cells in tumor‐bearing mice

Recent studies show that the liver is a preferred organ for the accumulation of MDSC. In this study, we examined the effect of systemic RB6‐8C5 treatment on hepatic MDSC in tumor‐bearing mice. EL4 tumor‐bearing mice were injected i.p. with RB6‐8C5, and hepatic, splenic, and blood MDSCs were analyzed by flow cytometry. Unexpectedly, hepatic MDSC remained in the liver, although RB6‐8C5 completely eliminated them from the spleen and peripheral blood 24 h after treatment. Secondary antibody staining confirmed the presence of RB6‐8C5‐bound MDSC in the liver of mice with s.c. tumors. Similar observations were made in two other (colon and melanoma) tumor models. Whereas RB6‐8C5 injection induced cell death of hepatic MDSC, as shown by Annexin V/7‐AAD staining, these cells were replaced immediately, leading to a constant, increased frequency of hepatic MDSC. Adoptively transferred MDSC migrated preferentially to the liver after RB6‐8C5 treatment, suggesting that hepatic MDSCs are reconstituted rapidly after depletion. Finally, hepatic MDSC remained immunosuppressive despite RB6‐8C5 injection. Our study demonstrates that RB6‐8C5 is not suitable for depletion of hepatic MDSCs and analysis of their function.

Regulation of monocyte cell fate by blood vessels mediated by Notch signalling

A population of monocytes, known as Ly6Clo monocytes, patrol blood vessels by crawling along the vascular endothelium. Here we show that endothelial cells control their origin through Notch signalling. Using combinations of conditional genetic deletion strategies and cell-fate tracking experiments we show that Notch2 regulates conversion of Ly6Chi monocytes into Ly6Clo monocytes in vivo and in vitro, thereby regulating monocyte cell fate under steady-state conditions. This process is controlled by Notch ligand delta-like 1 (Dll1) expressed by a population of endothelial cells that constitute distinct vascular niches in the bone marrow and spleen in vivo, while culture on recombinant DLL1 induces monocyte conversion in vitro. Thus, blood vessels regulate monocyte conversion, a form of committed myeloid cell fate regulation.

Peptidases released by necrotic cells control CD8+ T cell cross-priming

Cross-priming of CD8+ T cells and generation of effector immune responses is pivotal for tumor immunity as well as for successful anticancer vaccination and therapy. Dead and dying cells produce signals that can influence Ag processing and presentation; however, there is conflicting evidence regarding the immunogenicity of necrotic cell death. We used a mouse model of sterile necrosis, in which mice were injected with sterile primary necrotic cells, to investigate a role of these cells in priming of CD8+ T cells. We discovered a molecular mechanism operating in Ag donor cells that regulates cross-priming of CD8+ T cells during primary sterile necrosis and thereby controls adaptive immune responses. We found that the cellular peptidases dipeptidyl peptidase 3 (DPP-3) and thimet oligopeptidase 1 (TOP-1), both of which are present in nonimmunogenic necrotic cells, eliminated proteasomal degradation products and blocked Ag cross-presentation. While sterile necrotic tumor cells failed to induce CD8+ T cell responses, their nonimmunogenicity could be reversed in vitro and in vivo by inactivation of DPP-3 and TOP-1. These results indicate that control of cross-priming and thereby immunogenicity of primary sterile necrosis relies on proteasome-dependent oligopeptide generation and functional status of peptidases in Ag donor cells.

Tumor Induced Hepatic Myeloid Derived Suppressor Cells Can Cause Moderate Liver Damage

Subcutaneous tumors induce the accumulation of myeloid derived suppressor cells (MDSC) not only in blood and spleens, but also in livers of these animals. Unexpectedly, we observed a moderate increase in serum transaminases in mice with EL4 subcutaneous tumors, which prompted us to study the relationship of hepatic MDSC accumulation and liver injury. MDSC were the predominant immune cell population expanding in livers of all subcutaneous tumor models investigated (RIL175, B16, EL4, CT26 and BNL), while liver injury was only observed in EL4 and B16 tumor-bearing mice. Elimination of hepatic MDSC in EL4 tumor-bearing mice using low dose 5-fluorouracil (5-FU) treatment reversed transaminase elevation and adoptive transfer of hepatic MDSC from B16 tumor-bearing mice caused transaminase elevation indicating a direct MDSC mediated effect. Surprisingly, hepatic MDSC from B16 tumor-bearing mice partially lost their damage-inducing potency when transferred into mice bearing non damage-inducing RIL175 tumors. Furthermore, MDSC expansion and MDSC-mediated liver injury further increased with growing tumor burden and was associated with different cytokines including GM-CSF, VEGF, interleukin-6, CCL2 and KC, depending on the tumor model used. In contrast to previous findings, which have implicated MDSC only in protection from T cell-mediated hepatitis, we show that tumor-induced hepatic MDSC themselves can cause moderate liver damage.

Tumor‐induced CD11b+ Gr‐1+ myeloid‐derived suppressor cells exacerbate immune‐mediated hepatitis in mice in a CD40‐dependent manner

Immunosuppressive CD11b+Gr‐1+ myeloid‐derived suppressor cells (MDSCs) accumulate in the livers of tumor‐bearing (TB) mice. We studied hepatic MDSCs in two murine models of immune‐mediated hepatitis. Unexpectedly, treatment of TB mice with Concanavalin A (Con A) or α‐galactosylceramide resulted in increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) serum levels in comparison to tumor‐free mice. Adoptive transfer of hepatic MDSCs into naïve mice exacerbated Con A induced liver damage. Hepatic CD11b+Gr‐1+ cells revealed a polarized proinflammatory gene signature after Con A treatment. An IFN‐γ‐dependent upregulation of CD40 on hepatic CD11b+Gr‐1+ cells along with an upregulation of CD80, CD86, and CD1d after Con A treatment was observed. Con A treatment resulted in a loss of suppressor function by tumor‐induced CD11b+Gr‐1+ MDSCs as well as enhanced reactive oxygen species (ROS)‐mediated hepatotoxicity. CD40 knockdown in hepatic MDSCs led to increased arginase activity upon Con A treatment and lower ALT/AST serum levels. Finally, blockade of arginase activity in Cd40−/− tumor‐induced myeloid cells resulted in exacerbation of hepatitis and increased ROS production in vivo. Our findings indicate that in a setting of acute hepatitis, tumor‐induced hepatic MDSCs act as proinflammatory immune effector cells capable of killing hepatocytes in a CD40‐dependent manner.

Blood vessel control of macrophage maturation promotes arteriogenesis in ischemia

Ischemia causes an inflammatory response that is intended to restore perfusion and homeostasis yet often aggravates damage. Here we show, using conditional genetic deletion strategies together with adoptive cell transfer experiments in a mouse model of hind limb ischemia, that blood vessels control macrophage differentiation and maturation from recruited monocytes via Notch signaling, which in turn promotes arteriogenesis and tissue repair. Macrophage maturation is controlled by Notch ligand Dll1 expressed in vascular endothelial cells of arteries and requires macrophage canonical Notch signaling via Rbpj, which simultaneously suppresses an inflammatory macrophage fate. Conversely, conditional mutant mice lacking Dll1 or Rbpj show proliferation and transient accumulation of inflammatory macrophages, which antagonizes arteriogenesis and tissue repair. Furthermore, the effects of Notch are sufficient to generate mature macrophages from monocytes ex vivo that display a stable anti-inflammatory phenotype when challenged with pro-inflammatory stimuli. Thus, angiocrine Notch signaling fosters macrophage maturation during ischemia.Molecular mechanisms of macrophage-mediated regulation of artery growth in response to ischemia are poorly understood. Here the authors show that vascular endothelium controls macrophage maturation and differentiation via Notch signaling, which in turn promotes arteriogenesis and ischemic tissue recovery.

CD40 dependent exacerbation of immune mediated hepatitis by hepatic CD11b+ Gr-1+ myeloid derived suppressor cells in tumor bearing mice

Immunosuppressive CD11b+Gr‐1+ myeloid‐derived suppressor cells (MDSCs) accumulate in the livers of tumor‐bearing (TB) mice. We studied hepatic MDSCs in two murine models of immune‐mediated hepatitis. Unexpectedly, treatment of TB mice with Concanavalin A (Con A) or α‐galactosylceramide resulted in increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) serum levels in comparison to tumor‐free mice. Adoptive transfer of hepatic MDSCs into naïve mice exacerbated Con A induced liver damage. Hepatic CD11b+Gr‐1+ cells revealed a polarized proinflammatory gene signature after Con A treatment. An IFN‐γ‐dependent upregulation of CD40 on hepatic CD11b+Gr‐1+ cells along with an upregulation of CD80, CD86, and CD1d after Con A treatment was observed. Con A treatment resulted in a loss of suppressor function by tumor‐induced CD11b+Gr‐1+ MDSCs as well as enhanced reactive oxygen species (ROS)‐mediated hepatotoxicity. CD40 knockdown in hepatic MDSCs led to increased arginase activity upon Con A treatment and lower ALT/AST serum levels. Finally, blockade of arginase activity in Cd40−/− tumor‐induced myeloid cells resulted in exacerbation of hepatitis and increased ROS production in vivo. Our findings indicate that in a setting of acute hepatitis, tumor‐induced hepatic MDSCs act as proinflammatory immune effector cells capable of killing hepatocytes in a CD40‐dependent manner.

Corrigendum: Regulation of monocyte cell fate by blood vessels mediated by Notch signalling

Corrigendum: Regulation of monocyte cell fate by blood vessels mediated by Notch signalling